Physical Poster + E-Poster Presentation 34th Lorne Cancer Conference 2022

Determining the essential functions of distinct pro-survival BCL-2 family members by using gene-swap mice (#115)

Kerstin Brinkmann 1 , Andrew Kueh 1 , Marco Herold 1 , Anne Voss 1 , Andreas Strasser 1
  1. Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia

The pro-survival BCL-2 family member Mcl-1 is among the top 10 oncogenes that is amplified in human cancers1. Inhibitors of MCL-1 have recently entered clinical trials, however despite promising pre-clinical results, certain concerning side effects were observed1. Amongst the 5 mammalian pro-survival BCL-2 family members, that inhibit the mitochondrial apoptotic pathway, MCL-1 is unique as it is essential for early embryonic development and the survival of many cell types that are not impacted by the loss of any of the other pro-survival BCL-2 proteins. To date it remains unclear what characteristics of MCL-1 make it so unique. Notably, a non-apoptotic function of MCL-1 in mitochondrial structure and energy production was proposed. For the clinical testing and hopefully implementation of MCL-1 inhibiting drugs, it is important to know whether apoptosis-unrelated functions of MCL-1 do actually exist and, if so, whether they contribute to the toxicity to normal tissues - and/or efficacy against malignant cells - of MCL-1 inhibitors.

To identify the unique functions of MCL-1 we generated “gene-swap” mice by replacing its coding region with that for BCL-XL, BCL-2.  In contrast to the E3.5 embryonic lethality of Mcl-1-/- mice, homozygous Bcl-X>Mcl-1Ki/Ki and Bcl-2>Mcl-1Ki/Ki gene-swap mice developed until E12.5 and beyond on an imbred C57/BL6 background. The majority of homozygous gene swap embryos however was lost before E15.5. Embryos at earlier stages such as E11.5 and E13.5 show severe developmental abnormalities such as oedema, cranial bleedings, increased vascularisation and increased cellularity in several brain areas. We believe that this is caused by too much survival resulting from the replacement of an extremely short lived MCL-1 protein with the long lived BCl-XL or BCL-2 protein.

Exitingly, mice with a homozygous gene replacement (Bcl-x>Mcl-1Ki/Ki) that were bred on a mixed genetic background (FV/BalbC/C57/BL6), that is less susceptible to developmental apoptosis and abortion, were born. These mice are severely underdeveloped and die at around 25 days of age. Our preliminary analysis revealed that the mice suffered from severe liver damage demonstrated by ballooning hepatocytes, that might be the resut of impaired mitochondrial energy production.    

Thus, it appears that there is no unique apoptosis-unrelated function of MCL-1 that is essential for early embryogenesis. However, it might be possible that certain cell types that highly depend on mitochondrial energy production (e.g. hepatocytes) rely on an additional apoptosis-unrelated function of MCL-1 for their survival.